Pierce enhanced chemiluminescence ecl substrate

Cells were lysed at 48 hpt or 16 hpi in RIPA buffer (Abcam, Cambridge, UK) with cOmplete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland ) and PhosStop (Roche). Cell lysates were separated on a 10% polyacrylamide gel, followed by blotting on PVDF membrane (GE healthcare, Piscataway, NJ, USA). Regular blots were blocked in 5% nonfat milk diluted in 0.1% Tween-20 in PBS for 1 h at room temperature. When using phosphorylated protein-specific antibodies, 5% bovine serum albumin (MP Biomedicals, Santa Ana, USA) diluted in 0.1% Tween-20 in PBS was used for blocking. Primary antibodies were incubated overnight at 4 °C (Table 3). Following 3 consecutive 5 min washes in PBS-T, the membranes were incubated with the secondary antibody for 1h at room temperature. Following 3 more 5 min washes, the blots were detected using Pierce enhanced chemiluminescence (ECL) substrate (ThermoFisher, Waltham, MA, USA), ECL Plus substrate (GE Healthcare, Piscataway, NJ, USA), or SuperSignal West Femto maximum sensitivity substrate (ThermoFisher, Waltham, MA, USA) on a ChemiDoc MP imaging device (Bio-Rad, Hercules, CA, USA).

Plasma samples were collected from rGDF11 and vehicle-treated mdx mice, as previously described [14 (link)], and quantified with the Bradford regent (Sigma). Briefly, samples were diluted in Laemmli buffer and boiled for 10 min at 100 °C. Equal amounts (75 μg) of protein from each single mouse were loaded onto 4–20 % gradient mini-PROTEAN^® TGX Stain-Free™ gels (BioRad Labs). Proteins were transferred onto polyvinyl difluoride (PVDF) membranes (Millipore), which were then blocked in BSA 5 % Tris-buffered saline (TBS) with 0.05 % Tween-20 (TBST) for 1 h at RT. Membranes were incubated with rabbit anti-GDF11 antibody (1:1000, Abcam) overnight at 4 °C. The following day, these were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (GE healthcare). Proteins were detected by Pierce enhanced chemiluminescence (ECL) substrate (Thermo Scientific).

JeKo-1, Mino (parental/WT or BTK C481S mutant) and Granta cells were plated in 10 cm tissue culture dishes at a cell density of 2 x 10⁶ and were incubated overnight. The cells were serum starved for 6 h and then treated in the presence of increasing concentration of SRX3305 or Ibrutinib for 1 h, followed by stimulation with 10 μg/mL of goat F(ab) 2 anti-human antibody (Southern Biotech, Birmingham, Cat # 2022-01) for 15 min at 37 °C. Whole cell lysates of the treated and untreated JeKo-1 and Mino cells were prepared using RIPA buffer supplemented with protease, phosphatase/protease inhibitor cocktails (Thermo Scientific) and 0.1% NP40. Protein concentration in cell lysates was determined using a bicinchoninic acid (BCA) assay kit (Thermo Fisher). Equal amounts of lysate were resolved on a 4-12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with one or more of the following antibodies: anti-pBTK-Y223 (Cat # 5082S), anti-pAKT-S473 (Cat # 9271S), total anti-BTK (Cat # 8547S), total anti-AKT (Cat # 4691), and anti-β-actin (Cat # SC69879). Secondary antibodies were chosen according to the species of origin of the primary antibody. Protein bands were detected using the Pierce enhanced chemiluminescence (ECL) substrate (Thermo Fisher) and imaging system. Uncropped western blots images are shown in Supplementary Fig. 7.